Hydroxyproline-rich proteins and pharmaceutical and cosmetic formulations containing them

ABSTRACT

PCT No. PCT/EP95/05084 Sec. 371 Date Jun. 18, 1997 Sec. 102(e) Date Jun. 18, 1997 PCT Filed Dec. 21, 1995 PCT Pub. No. WO96/20284 PCT Pub. Date Jul. 4, 1996The invention relates to hydroxyproline-rich glycoproteins, which can be obtained by acid alcohol extraction from Taxus supp., Gingko biloba, Lycopersicum esculentum and Daucus carota cell cultures, having the following characteristics: average molecular weight 20,000 Daltons with variability interval 12,000 to 38,000, determined by means of gel permeation and electrophoresis; high solubility in water.

TECHNICAL FIELD

The present invention relates to hydroxyproline-rich glycoproteins whichcan be obtained from vegetable sources, and to the pharmaceutical andcosmetic use thereof.

More precisely, the invention relates to hydroxyproline-richglycoproteins, which can be obtained by acid alcohol extraction fromTaxus spp., Gingko biloba, Lycopersicum esculentum and Daucus carotacell cultures, having the following characteristics:

average molecular weight 20,000 Daltons with variability interval 12,000to 38,000, determined by means of gel permeation and electrophoresis;

solubility in acid aqueous solutions.

Some glycoproteins of animal origin, such as collagen and proteoglycans,are known to exert a beneficial action on the skin when appliedtopically as such or incorporated in suitable formulations.

BACKGROUND ART

Collagen, which is a glycoprotein rich in proline and hydroxyproline, isespecially used as such or combined with other polypeptide bases in thetreatment of wrinkles and other unaesthetic blemishes linked to poorskin hydration and elasticity. The animal origin of collage, however,limits its use because of the risks of contamination from viruses andtoxins. Though the compounds of vegetable origin do not involve theserisks, so far their use in cosmetics has been quite limited: forexamples, cosmetic formulations are known which contain raw extracts ofsuch plants as Aloe or even entire minced vegetables such as avocado.

Vegetable glycoproteins, called extensines, that are produced fromvegetable cells in the proliferation stage and have a similar structureto animal collagen, are known. EP-A-0 533 4078 discloses the cosmeticuse of extensines having an average molecular weight above 100,000Daltons. However, the methods for the extraction of extensines describedto date, which involve the extraction of vegetable materials of variousorigin by means of aqueous saline solutions, followed by purificationwith strong acids such as trichloroacetic acid, do not allow to obtainsuitable products for cosmetics, due to problems concerning solubility,stability, repeatability and consistency of their chemical-physicalcharacteristics.

SUMMARY OF THE INVENTION

It has now been found that it is possible to obtain hydroxyproline-richglycoproteins, structurally similar to the above described extensinesbut with a lower molecular weight and a higher solubility in acidaqueous solutions, by means of a procedure comprising the in vitroculture of cells of selected plants and the extraction, with acidalcoholic solutions, of the cells grown in a suitable medium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The glycoproteins obtainable according to the invention have hydrating,film-forming, toning and cicatrizant properties higher than those ofcollagen. The glycoproteins of this invention can therefore be employedin cosmetic or dermatologic formulations for the treatment of dry skin,psoriasis, ichtyosis, dandruff, keratosis, wrinkles, acne, eczema,inflammatory dermatosis, ageing of the skin and all the otherapplications for which the use of animal collagen has been proposed.

The aqueous solutions of the glycoproteins of the invention remainstable without any polymerisation of the glycoproteins leading to theformation of insoluble products. In addition, the viscosity of thesesolutions is particularly high and not dependent on the concentrations;0.1% concentrations surprisingly have the same film-forming andhydrating power equal as 1% collagen or 5% vegetable albumin solutions.

The vegetable material to be extracted is obtained from fermentercultures of Taxus spp., Gingko biloba, Lycopersicum esculentum andDaucus carota cells. The use of cells from the species Taxus spp.,Gingko biloba and Lycopersicum esculentum is particularly preferred. Thecell culture techniques are conventional and include the suspensionculture starting from callus cultures from various parts of the plantssuch as leaves, bark, roots, trunk or seeds, as described by Dobbs andRoberts, Experiments in Plant Tissue Culture, 2nd ed. CambridgeUniversity Press, New York, 1985.

The vegetable tissue of the callus, following sterilisation and optionaladdition of antibacterials, is typically used for the inoculum ofsuitable liquid culture media as described in the above mentioned Manualby Dobbs and Roberts. A particularly suitable medium for this inventionis the Murashige and Skoog medium. The addition of specific additivessuch as proline, reducing agents, ethylene or compounds capable ofreleasing ethylene such as Ethephon or L-aminocyclo-propanecarboxylicacid, may be suitable to increase productivity in the desiredglycoproteins.

The use of naphthylacetic acid as the as auxin,6-(γ,γ-dimethylamino)-purine as the cytokinin, vitamins and 3%saccharose as the carbon source is preferred. The addition of vitamin Cmay be suitable, depending on the material chosen, to prevent the finalproduct from browning.

The fermentation time may vary from 3 to 12 days and is preferablybetween 5 and 6 days. Once the fermentation has been completed, theculture medium is centrifuged and the cellular mass is extracted bymeans of alcohols, preferably ethanol, in the presence of dilutedmineral acids, preferably hydrochloric or sulphuric acid. This procedureinactivates some enzymes that may jeopardise the stability of theglycoproteins of the invention, specifically of polyphenoloxidase andtyrosine oxidase which favour the polymerization of glycoproteins withthe consequent formation of insoluble products.

The alcohol extraction in the presence of mineral acids allows thecomplete extraction of basic glycoproteins and has proved to beextremely selective to this end. Other water-mixable alcohols, such asmethanol or isopropanol, can be used besides ethanol. The resultinghydroalcoholic extracts are neutralised and then concentrated and heatedto a temperature of 70° C. to 100° C., preferably around 80° C., up tocomplete precipitation of the denatured proteins. The suspension is thenclarified by concentration and the fluid is subjected to fractionalultrafiltration to remove high and low molecular weight substances.Ultrafiltration is performed by means of polysulphonic membranes havingcut-off of 10,000 Daltons to 40,000 Daltons, such as Centricon^(R) orRomicon^(R), whose fibres may be hollow or, alternatively, coiled. Theresulting filtered product is electrodialysed to remove undesiredsubstances such as salts and low molecular weight sugars. Afterfiltration and dialysis, the resulting solution can be used as such incosmetic or pharmaceutical preparations or it can be concentrated to alower volume and then lyophilised or atomised.

The analytical characterization of the products of the invention wascarried out by gel permeation using a high-pressure liquid chromatographconsisting of a Waters pump unit and provided with a UltrahydrogelLinear Waters^(R) column battery 30 cm×0.5 cm and Waters UV absorptiondetector, model 484. An aqueous solution containing 0.067 Mmonopotassium phosphate, 0.1 M NaCl and 6×10⁻⁴ M NaN₃ was used as theeluent. The glycoprotein samples to be analysed are dissolved in thesame eluent solution (3 mg/10 ml) and scalar amounts of the substance aswell as the reference substances selected as molecular weights betweenCytochrome C (12,400 Daltons) and dextran blue (2,000,000 Daltons);alternatively or simultaneously the products or their intermediates canbe determined by electrophoresis on 12.5% polyacrylamide gel and 4%stacking gel. The samples to be analysed are dissolved in a buffercontaining SDS and 0.1% mercaptoethanol while depositing quantitiesbetween 100 mg and 300 mg. The migration is carried out at a constantcurrent at 20 mA for 4 hours. A gauging curve is drawn with 5 standardweights (7 kD, 14 kD, 24 kD, 54 kD and 66 kD). Weights of 22.5 kD and 25kD are calculated from this gauging curve for the two main bands andweights of 31 kD and 34 kD are calculated for the less intense bands.The procedures described here allow mixtures of products with comparablemolecular weights and comparable amino acid compositions to be obtainedfrom the various cell explants starting from different plants. Theresults of the amino acid analysis of glycoproteins extracted fromGinkgo biloba cells are shown below as an example.

    ______________________________________                                        Amino acid    Peak area %                                                     ______________________________________                                        Asp           4.399                                                           Glu           4.328                                                           Hyp           17.505                                                          Ser           7.065                                                           Gly           6.056                                                           Hys           1.782                                                           Arg           2.471                                                           Thr           4.739                                                           Pro           10.036                                                          Ala           8.2                                                             Tyr           2.388                                                           Val           6.162                                                           Met           1.154                                                           Ile           2.479                                                           Leu           5.525                                                           Phe           1.862                                                           Lys           14.254                                                          ______________________________________                                    

The above data refer to the percentage of the total amount of aminoacids present in the glycoprotein mixture. The sugars in the mixture arearabinose and galactose. The ratio of amino acids to sugars is onaverage 2:1 for the various products.

As mentioned above, the products according to the invention can be usedboth in the pharmaceutical and cosmetic fields. For the pharmaceuticalfield, the product may be incorporated in gels or ointments or appliedon medicated gauzes for specific treatment of burns or wounds. In thiscase the product is usually subjected to sterilisation or sterilefiltrations and lyophilised.

The cosmetic and dermatologic preparations of the invention can beprepared according to traditional methods. Examples of administrationforms include aqueous sprays, lotions, solutions, emulsions, gels,ointments and creams.

The cosmetic and dermatologic preparations of the inventions can containhydroxyproline-rich glycoproteins in weight percentages of about 0.01%to about 50%, preferably from 0.05% to 5%, as well as conventionalexcipients. Given the high stability of the glycoproteins of thisinvention, pharmaceutical and cosmetic preparations containing above 50%of soluble hydroxyproline-rich glycoproteins can be obtained.

The glycoproteins of the invention can be added to pharmaceutical andcosmetic preparations as such or microencapsulated so as to provide along-term hydrating action. The microcapsules can be either hydrophilicor lipophilic. The preparations of the invention may include otheractive principles having complementary or useful activity for thedesired aims.

EXAMPLES

The invention is further illustrated by the following examples.

Example 1

Preparation of the callus and liquid culture of Ginkgo biloba for theproduction of glycoproteins

An explant of young leaves of Ginkgo biloba is prepared by washing theleaves in a 0.1% Tween ₈₀.sup.(R) solution. The laminae are sectioned infractions of about 0.5 cm and pre-sterilised for 1 minute with 75%ethanol. The sterilisation is then completed with a 2% sodiumhypochlorite solution and triple washing of the explant in sterilewater. The resulting explants are transferred to a Petri dish inMurashige & Skoog medium containing 3% saccharose with the addition ofLynsmeyer & Skoog vitamins and hormones such as2,4-dichlorophenoxyacetic acid and naphthylacetic acid. The products areincubated in the dark at 23° C. for 20 days. At the end of this period,friable calli are obtained which grow easily and are moved in continuousrows by means of subcultures in the same conditions, as they can be usedfor propagation in a liquid medium. These calli are used to inoculateErlenmeyer flasks containing 200 ml of Murashige & Skoog medium, withthe addition of naphthylacetic acid and 6 (γ,γ-dimethylamino)-purine,Lynsmeyer & Skoog vitamins and 3% saccharose as a source of carbon. Theflasks are incubated with stirring in continuous light for 4 days, afterwhich the cell biomass is harvested for the extraction of glycoproteins.

Example 2

Preparation of glycoproteins from Ginkgo biloba cells

5 liters of the culture obtained according to Example 1 are low-speedcentrifuged and the harvested cells (1.5 kg of fresh weight) areextracted with 1.5 l of 70% ethanol containing 1% sulphuric acid. Theextraction is repeated twice thereby quantitatively recovering the basicglycoproteins. After neutralisation, the extracts are filtered to removeany turbidity and concentrated under vacuum at 50° C. until ethanol iscompletely removed. The aqueous concentrate is heated at 85° C. for 30minutes and centrifuged again to remove the precipitate, which isdiscarded. The resulting clear solution is ultrafiltered by means of aCentricon^(R) membrane with cut-off 40,000 Dalton limit to exclude thehigher molecular weights.

The filtered product is then ultrafiltered using a hollow-fibre membranewith cut-off 10,000 Dalton to remove non-glycoprotein, low-molecularweight substances. The filtrate is then subjected to dialysis andconcentrated to 1% of solid residue. 1.5 liters of a slightly viscousproduct is obtained, which may be used as such in cosmetic formulations.At the electrophoresis analysis, the product contained 6 bands, 4 ofwhich had molecular weights of 16,000, 22,000, 33,000 and 36,000Daltons.

Example 3

Preparation of glycoproteins from Lycopersicum esculentum

Following the procedure of Example 1, a cell mass from sterile buds ofLycopersicum esculentum is prepared in a 14-liter fermenter containing10 liters of Murashige & Skoog medium added with naphthylacetic acid and6 (γ,γ-dimethylamino)-purine, Lynsmeyer & Skoog vitamins and 3%saccharose as a carbon source. The fermentation is carried on for 5 daysat 23° C. while stirring at 150 rpm in the presence of yeast extract at0.05% concentration and with an approximately 70% concentration ofdissolved oxygen. At the end of the fermentation the broth is gatheredand micro-filtered through a 0.2 μm ceramic membrane to concentrate thecells. Some isopropanol containing 0.5% hydrochloric acid is added tothe cell paste thus obtained and the method described in Example 2 isapplied to the extracts. 3.5 liters of a solution are obtained with 0.5%dry residue. The analysis of the lyophilised solutions gave a content of10% proline and 31% hydroxyproline, respectively.

Example 4

    ______________________________________                                        Cosmetic formulation                                                          ______________________________________                                        100 g of O/W emulsion contain:                                                SOLUTION OF THE EXAMPLE 2 OR 3                                                                         10.0 g                                               Acetylated lanolin alcohol PEG-10                                                                       2.0 g                                               Cetyl-stearyl alcohol     1.5 g                                               Cetyl palmitate           2.0 g                                               Stearic acid              7.0 g                                               Octyl octanoate           7.5 g                                               Potassium cetyl phosphate                                                                               0.5 g                                               Preservatives            q.s.                                                 Fragrance                q.s.                                                 Purified water           q.s. to 100 g                                        ______________________________________                                    

Example

    ______________________________________                                        Cosmetic formulation                                                          ______________________________________                                        100 g of O/W emulsion contain:                                                SOLUTION OF THE EXAMPLE 2 OR 3                                                                         10.0 g                                               Cetyl stearyl glucoside   5.0 g                                               Jojoba oil               10.0 g                                               Isopropyl myristate       8.0 g                                               Dimethicone               0.5 g                                               Antioxidant              q.s.                                                 Preservatives            q.s.                                                 Fragrance                q.s.                                                 Purified water           q.s. to 100 g                                        ______________________________________                                    

What is claimed is:
 1. A method for obtaining hydroxyproline-richglycoproteins which comprises:a) culturing cells of Gingko biloba orLycopersicum esculentum in a liquid culture medium for 3 to 12 days; b)extracting the cultured cells with an alcohol having one to three carbonatoms in the presence of a dilute acid material to obtain an extract; c)neutralizing, concentrating and heating the extract at a temperature ofbetween 70° C. and 100° C.; and d) centrifuging the heated extract toobtain a supernatant and precipitate, discarding the precipitate, andsubjecting the supernatant to fractional ultrafiltration and dialysis toobtain the hydroxyproline-rich glycoproteins.
 2. Hydroxyproline-richglycoproteins prepared by the process of claim 1, wherein saidglycoproteins have an average molecular weight of 20,000 Daltons with avariable interval of 12,000 to 38,000 Daltons, as determined by SDS-Pageunder reducing conditions.
 3. The hydroxyproline-rich glycoproteins ofclaim 2, wherein the glycoproteins are obtained from cell cultures ofGingko biloba or Lycopersicum esculentum.
 4. A cosmetic orpharmaceutical preparation containing the hydroxyproline richglycoproteins prepared by the process of claim
 1. 5. A preparationaccording to claim 4 in the form of a lotion, solution, emulsion, gel,ointment, cream, or medicated gauze.
 6. The method of claim 1 whereinthe alcohol is methanol, ethanol or isopropanol.
 7. The method of claim1 wherein the cells are cultured for 5 to 6 days and the extract isheated to a temperature of about 80° C.